title: Molecular engineering of a fluorescent bioprobe for sensitive and selective detection of amphibole asbestos.
abstract: Fluorescence microscopy-based affinity assay could enable highly sensitive and selective detection of airborne asbestos, an inorganic environmental pollutant that can cause mesothelioma and lung cancer. We have selected an Escherichia coli histone-like nucleoid structuring protein, H-NS, as a promising candidate for an amphibole asbestos bioprobe. H-NS has high affinity to amphibole asbestos, but also binds to an increasingly common asbestos substitute, wollastonite. To develop a highly specific Bioprobe for amphibole asbestos, we first identified a specific but low-affinity amosite-binding sequence by slicing H-NS into several fragments. Second, we constructed a streptavidin tetramer complex displaying four amosite-binding fragments, resulting in the 250-fold increase in the probe affinity as compared to the single fragment. The tetramer probe had sufficient affinity and specificity for detecting all the five types of asbestos in the amphibole group, and could be used to distinguish them from wollastonite. In order to clarify the binding mechanism and identify the amino acid residues contributing to the probe's affinity to amosite fibers, we constructed a number of shorter and substituted peptides. We found that the probable binding mechanism is electrostatic interaction, with positively charged side chains of lysine residues being primarily responsible for the probe's affinity to asbestos.
authors.affiliation: Department of Molecular Biotechnology, Higashihiroshima, Hiroshima, Japan.
authors.forename: Takenori
authors.initials: T
authors.lastname: Ishida
authors.forename: Maxym
authors.initials: M
authors.lastname: Alexandrov
authors.forename: Tomoki
authors.initials: T
authors.lastname: Nishimura
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Hirota
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ikeda
authors.forename: Akio
authors.initials: A
authors.lastname: Kuroda
mesh_terms: D000393:Air Pollutants / Q000302:isolation & purification*; D017636:Asbestos, Amphibole / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D057005:Bioengineering / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D012680:Sensitivity and Specificity; D055672:Static Electricity; D019809:Streptavidin / Q000378:metabolism
chemical_list: D000393:Air Pollutants; D017636:Asbestos, Amphibole; D029968:Escherichia coli Proteins; C529458:FimG protein, E coli; D036781:Fimbriae Proteins; D019809:Streptavidin